This application relates to a receptor protein tyrosine kinase ligand and its uses. In particular this application relates to the production and use of purified forms of AL-2 and related proteins.
Protein neurotrophic factors, or neurotrophins, which influence growth and development of the vertebrate nervous system, are believed to play an important role in promoting the differentiation, survival, and function of diverse groups of neurons in the brain and periphery. Neurotrophic factors are believed to have important signaling functions in neural tissues, based in part upon the precedent established with nerve growth factor (NGF). NGF supports the survival of sympathetic, sensory, and basal forebrain neurons both in vitro and in vivo. Administration of exogenous NGF rescues neurons from cell death during development. Conversely, removal or sequestration of endogenous NGF by administration of anti-NGF antibodies promotes such cell death (Heumann, J. Exp. Biol., 132:133-150 (1987); Hefti, J. Neurosci., 6:2155-2162(1986); Thoenen et al., Annu. Rev. Physiol., 60:284-335 (1980)).
Additional neurotrophic factors related to NGF have since been identified. These include brain-derived neurotrophic factor (BDNF) (Leibrock, et al., Nature, 341:149-152 (1989)), neurotrophin-3 (NT-3) (Kaisho, et al., FEBS Lett., 266:187 (1990); Maisonpierre, et al., Science, 247:1446 (1990); Rosenthal, et al., Neuron, 4:767 (1990)), and neurotrophin 4/5 (NT-4/5) (Berkmeier, et al., Neuron, 7:857-866 (1991)).
Neurotrophins, similar to other polypeptide growth factors, affect their target cells through interactions with cell surface receptors. According to our current understanding, two kinds of transmembrane glycoproteins act as receptors for the known neurotrophins. Equilibrium binding studies have shown that neurotrophin-responsive neuronal cells possess a common low molecular weight (65,000-80,000 Daltons), a low affinity receptor typically referred to as p75LNGFR or p75, and a high molecular weight (130,000-150,000 Dalton) receptor. The high and low affinity receptors are members of the trk family of receptor tyrosine kinases.
Receptor tyrosine kinases are known to serve as receptors for a variety of protein factors that promote cellular proliferation, differentiation, and survival. In addition to the trk receptors, examples of other receptor tyrosine kinases include the receptors for epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF). Typically, these receptors span the cell membrane, with one portion of the receptor being intracellular and in contact with the cytoplasm, and another portion of the receptor being extracellular. Binding of a ligand to the extracellular portion of the receptor induces tyrosine kinase activity in the intracellular portion of the receptor, with ensuing phosphorylation of various intracellular proteins involved in cellular signaling pathways.
Recently, a receptor tyrosine kinase subclass referred to as the Eph receptor subclass or family has been identified. Eph was the first member of this Eph subclass of receptor tyrosine kinases to be identified and characterized by molecular cloning (Hirai et al., Science, 238:1717-1720 (1987)). The name Eph is derived from the name of the cell line from which the Eph cDNA was first isolated, the erythropoietin-producing human hepatocellular carcinoma cell line, ETL-1. The general structure of Eph is similar to that of other receptor tyrosine kinases and consists of an extracellular domain, a single membrane spanning region, and a conserved tyrosine kinase catalytic domain. However, the structure of the extracellular domain of Eph, which comprises an immunoglobulin (Ig)-like domain at its amino terminus, followed by a cysteine-rich region and two fibronectin type III repeats in close proximity to the transmembrane domain, is completely distinct from that of previously described receptor tyrosine kinases. The juxtamembrane domain and carboxy-terminus regions of Eph also are unrelated to the corresponding regions of other tyrosine kinase receptors.
Newly discovered members of the Eph receptor family include Elk, Cek5, Mek4, Cek4, Hek/Hek4 (Sajjadi et al., The New Biologist, 3:769-778 (1991)), Cek6 through Cek10 (Sajjadi et al., Oncogene, 8:1807-13 (1993), Sek, Hek2, and Ehk3 (Tuzi, et al., Br. J. Cancer, 69:417-421 (1994); Zhou, et J. Neurosci. Res., 37:129-143 (1994)). Other Eph-related receptor kinases that have been identified include Sek (Gilardi-Hebenstreit et al, Oncogene, 7:2499-2506 (1992)), Eck (Lindberg et al., Mol. Cell. Biol., 10:6316-6324 (1990)), Elk (Lhotak et al., Mol. Cell. Biol., 11:2496-2502 (1991)), Eek (Chan et al., Oncogene, 6:1057-1061 (1991)), Rek7 (Winslow et al., Neuron, 14:973-981 (1995). Rek7 is a rat homolog of chicken Cek7 and closely related to Ehk-1 (Davis et al., Science, 266:816-819 (1994)) and bsk (Maisonpierre et al., Oncogene, 8:3277-3288 (1993); Zhou et al., J. Neurosci. Res,. 37:129-143 (1994)); the Rek7 cDNA corresponds to a splice variant of Ehk-1, lacking the first of two tandem fibronectin type-III domains. Human homologs of the chicken Cek receptors are referred to as Hek receptors (See WO 95/28484, which is incorporated herein by reference). For example, Hek5 (Fox et al., Oncogene, 10(5):897-905 (1995); WO 95/28484) is the human homolog of chicken Cek5. The amino acid sequence of Hek5 is very closely related (96% amino acid identity in the catalytic domain) to the chicken receptor Cek5 (Pasquale et al., J. Neuroscience, 12:3956-3967 (1992); Pasquale, Cell Regulation, 2:523-534 (1991)). A portion of the Hek5 sequence was previously disclosed as Erk, a human clone encoding about sixty amino acids (Chan et al., Oncogene, 6:1057-1061 (1991)). Mature Erk showed high homology with Cek5 (92.5%) and mouse Nuk (99.1%) (Kiyokawa et al., Cancer Res., 54 (14):3645-50 (1994)). Other human Eph-family receptors include Hek (Wicks et al., Proc. Natl. Acad. Sci. USA, 89(5):161 1-1615 (1992); also known as Hek4), Hek2 (Bohme et al., Oncogene, 8:2857-2862 (1993)), Heks 7, 8 and 11 (WO 95/28484), Hek3, which is a homolog of rat Eek and murine Mdk-1, and Hek 12, which is a homolog of rat Ehk2.
Many of the Eph-receptor family members are xe2x80x9corphan receptors.xe2x80x9d However, recently, ligands have been reported including B61, an Eck receptor ligand (Bartley et al., Nature, 368:558-560 (1994) and Pandey et al., Science, (1995) 268:567-569), Elf-1, a Mek4 and Sek receptor ligand (Cheng et al., Cell, (1995) 82:371-381; Cheng et al., Cell, 79:157-168 (1994)), Htk-L (Bennett et al., Proc. Natl. Acad. Sci. USA, 92(6):1866-70 (1995)), AL-1 (Winslow et al, Neuron, 14:973-981 (1995)) and RAGS (Drescher et al., Cell, (1995) 82:359-370), which are Rek7 ligands, Ehk-1-L (Davis et al., Science, 266:816-819 (1994); see also efl-2 in WO 95/27060), Cek5-L, and Lerk2 (Beckmann et al., EMBO J., 13:3757-3762 (1994)).
Aberrant expression of receptor tyrosine kinases correlates with transforming ability. This relationship includes members of the Eph subclass of receptor tyrosine kinases. For example, carcinomas of the liver, lung, breast and colon show elevated expression of Eph. Unlike many other tyrosine kinases, this elevated expression can occur in the absence of gene amplification or rearrangement. Such involvement of Eph in carcinogenesis also has been shown by the formation of foci of NIH 3T3 cells in soft agar and of tumors in nude mice following overexpression of Eph. Moreover, Hek has been identified as a leukemia-specific marker present on the surface of a pre-B cell leukemia cell line. As with Eph, Hek also was overexpressed in the absence of gene amplification or rearrangements in, for example, hemopoietic tumors and lymphoid tumor cell lines. Over-expression of Myk-1 (a murine homolog of human Htk (Bennett et al., J. Biol. Chem., 269(19): 14211-8 (1994)) was found in the undifferentiated and invasive mammary tumors of transgenic mice expressing the Ha-ras oncogene. (Andres et al., Oncogene, 9(5): 1461-7 (1994) and Andres et al., Oncogene, 9(8):2431 (1994)).
In addition to their roles in carcinogenesis, a number of transmembrane tyrosine kinases have been reported to play key roles during development. Some receptor tyrosine kinases are developmentally regulated and predominantly expressed in embryonic tissues. Examples include Cek1, which belongs to the FGF subclass, and the Cek4 and Cek5 tyrosine kinases (Pasquale et al., Proc. Natl. Acad Sci., USA, 86:5449-5453 (1989); Sajjadi et al., New Biol., 3(8):769-78 (1991); and Pasquale, Cell Regulation, 2:523-534 (1991)).
Eph family members are expressed in many different adult tissues, with several family members expressed in the nervous system or specifically in neurons (Maisonpierre et al., Oncogene, 8:3277-3288 (1993); Lai et al., Neuron, 6:691-704(1991)).
The aberrant expression or uncontrolled regulation of any one of these receptor tyrosine kinases can result in different malignancies and pathological disorders. Therefore, there exists a need to identify means to regulate, control and manipulate receptor tyrosine kinases and their ligands in order to provide new and additional means for the diagnosis and therapy of Eph-pathway related disorders and cellular processes. The present application provides the clinician and researcher with such means by providing new molecules that are specific for interacting with Eph-family receptors. These compounds and their methods of use, as provided herein, allow exquisite therapeutic control and specificity. Additional advantages are provided as well.
The present invention provides a novel cytokine, an Eph-related tyrosine kinase receptor ligand referred to as AL-2.
The present invention provides nucleic acid encoding AL-2, particularly two forms referred to herein as AL-2s (xe2x80x9cAL-2-shortxe2x80x9d) and AL-21 (xe2x80x9cAL-2-longxe2x80x9d), and methods to use the nucleic acid to produce AL-2 in recombinant host cells for diagnostic or therapeutic purposes. Also provided are uses of nucleic acids encoding AL-2, and portions thereof, to identify related nucleic acids in the cells or tissues of various animal species.
By providing the full nucleotide coding sequence for AL-2, the invention enables the production of AL-2 by means of recombinant DNA technology, thereby making available for the first time sufficient quantities of substantially pure AL-2 protein or AL-2 antagonists for diagnostic and therapeutic uses. For example, method embodiments include treatment or prevention of a variety of neurological disorders and diseases as well as conditions that are angiogenesis-dependent such as solid tumors, diabetic retinopathy, rheumatoid arthritis, and wound healing.
Also provided are derivatives and modified forms of AL-2, including amino acid sequence variants and covalent derivatives thereof, as well as antagonists of AL-2, that are preferably biologically active (e.g., antigenically active. In one embodiment, the invention provides a soluble form of the ligand with at least the transmembrane region deleted. Usually, the cytoplasmic domain will also be absent. Immunogens are provided for raising antibodies, as well as to obtain antibodies, capable of binding to, preferably neutralizing, AL-2 or derivatives or modified forms thereof.
In a preferred embodiment, the invention provides AL-2 that is free of other human proteins.
AL-2 and modified and variant forms of AL-2 are produced by means of chemical or enzymatic treatment or by means of recombinant DNA technology, including in vivo production. Variant polypeptides can differ from native AL-2, for example, by virtue of one or more amino acid substitutions, deletions or insertions, or in the extent or pattern of glycosylation, but will substantially retain a biological activity of native AL-2.
Chimeras comprising AL-2 (or a portion thereof) fused to another polypeptide are provided. An example of such a chimera is epitope-tagged AL-2. In another embodiment a soluble form of an AL-2 chimera is provided, for example, as an immunoadhesin, which is a fusion of the extracellular domain of AL-2 and an immunoglobulin sequence.
Antibodies to AL-2 are produced by immunizing an animal with AL-2 or a fragment thereof, optionally in conjunction with an immunogenic polypeptide, and thereafter recovering antibodies from the serum of the immunized animals. Alternatively, monoclonal antibodies are prepared from cells of the immunized animal in conventional fashion. Antibodies obtained by routine screening will bind to AL-2 but, preferably, will not substantially bind to (i.e., cross react with) NGF, BDNF, NT-3, NT-4/5, GDNF, AL-1, Htk-L, Lerk-2, or other neurotrophic factors or cytokines; Immobilized anti-AL-2 antibodies are particularly useful in the detection of AL-2 in clinical samples for diagnostic purposes, and in the purification of AL-2.
AL-2, its derivatives, or its antibodies are formulated with physiologically acceptable carriers, especially for therapeutic use. Such carriers are used, for example, to provide sustained-release formulations of AL-2.
In further aspects, the invention provides a method for determining the presence of a nucleic acid molecule encoding AL-2 in test samples prepared from cells, tissues, or biological fluids, comprising contacting the test sample with isolated DNA comprising all or a portion of the nucleotide coding sequence for AL-2 and determining whether the isolated DNA hybridizes to a nucleic acid molecule in the test sample. DNA comprising all or a portion of the nucleotide coding sequence for AL-2 is also used in hybridization assays to identify and to isolate nucleic acids sharing substantial sequence identity to the coding sequence for AL-2, such as nucleic acids that encode allelic variants of AL-2.
Also provided is a method which involves contacting an AL-2 receptor with AL-2 in order to cause phosphorylation of the kinase domain of the receptor.
Also provided is a method for amplifying a nucleic acid molecule encoding AL-2 that is present in a test sample, comprising the use of an oligonucleotide having a portion of the nucleotide coding sequence for AL-2 as a primer in a polymerase chain reaction.